This application relates to the use of lysostaphin in the treatment and prevention of staphylococcal infection and, in particular, to the treatment and prevention of staphylococcal bovine mastitis.
Lysostaphin is a bacteriocin secreted by a single known strain of Staphylococcus simulans originally isolated and named Staphylococcus staphylolyticus by Schindler and Schuhardt. The production of lysostaphin by S. staphylolyticus has been described previously in U.S. Pat. No. 3,278,378 issued Oct. 11, 1966 and in Proceedings of the National Academy of Sciences, Vol. 51, pp. 414-421 (1964). The single organism S. staphylolyticus (NRRL B-2628) which produced lysostaphin was recently identified as a biovar of S. simulans by Sloan et al., Int. J. System. Bacteriol., Vol. 32, pp. 170-174 (1982). Since the name S. staphylolyticus is not on the Approved List of Bacterial Names, the organism producing lysostaphin has been redesignated as S. simulans.
Bacteriocins are proteins secreted by bacteria that kill and sometimes lyse related bacteria. For example, lysostaphin lyses and kills practically all known staphylococcal species but is inactive against bacteria of all other genera. Lysostaphin, isolated from culture filtrates of S. simulans (NRRL B-2628) grown according to published references, is an endopeptidase which cleaves the polyglycine cross-links of the peptidoglycan found in the cell walls of staphylococci. In addition, cultures that produce lysostaphin appear to be resistant to its activities while cultures grown under non-lysostaphin producing conditions are sensitive.
Previous studies have shown that lysostaphin can be produced by fermentation techniques wherein S. simulans is grown in liquid culture. Such fermentation techniques are described in U.S. Pat. No. 3,278,378 issued Oct. 11, 1966 and in Proceedings of the National Academy of Sciences, Vol. 51, pp. 414-421 (1964). Various improvements in the production of lysostaphin by fermentation techniques have also been made as documented in U.S. Pat. Nos. 3,398,056, issued Aug. 20, 1968, and 3,594,284, issued Jul. 20, 1971. The latter two references disclose improvements to culture medium and inoculation techniques whereby the production of lysostaphin by fermentation can be accelerated and improved. Lysostaphin is produced by S. simulans during exponential growth as an inactive precursor. The proenzyme is converted to active mature enzyme by protease produced by stationary phase cultures of S. simulans.
In addition, lysostaphin can be produced by recombinant microorganisms, including strains of E. coli, Bacillus subtilis and B. sphaericus which express the lysostaphin gene. In contrast to the natural production, lysostaphin accumulates during exponential growth in the culture medium of recombinant lysostaphin producing strains as fully processed mature active enzyme and is free of staphylococcal immunogenic contaminants.
Bovine mastitis is a costly problem to the dairy industry, costing over $2 billion per year in the United States alone. The disease is estimated to affect 50 percent of American dairy cows to some degree, leading to unusable milk, decreased milk production, and, in cases of severe infection, the death of the animal.
Mastitis is caused by infection of the milk glands, principally by Staphylococcus aureus or Streptococcus agalactiae, and to a lesser degree by E. coli and other gram-negative bacteria or combinations thereof. Most streptococcal infections have proven to be effectively treatable using conventional antibiotic therapy. Staphylococcal mastitis has, however, proven more difficult to cure.
Traditional prevention of bovine mastitis can involve a complex regimen of daily teat-dipping with a disinfectant solution, (See, J. S. McDonald, 6 Veterinary Clinics of North America Large Animal Practice 269 (1984)) and may, in some cases, involve antibiotic-containing teat dips. Routine antibiotic therapy must be approached with caution, however, to minimize selection for antibiotic resistant strains. When infection does occur, intramammary infusion of antibiotics is indicated. Antibiotic therapy of this kind can reduce the infection so that the milk produced is saleable, but it generally does not lead to complete elimination of the causative organism.
In the past, staphylococcal mastitis has shown a poor response to antibiotic therapy and a tendency for infections to recur and become chronic. Studies on mastitis have indicated that part of the problem in treating mastitis is that a significant number of staphylococci remain viable in the mammary gland within phagocytic polymorphonuclear neutrophil leukocytes (PMN). It is believed that the staphylococci within the PMN are protected from the effects of the antibiotic, and, when lysis of the leukocyte occurs, the phagocytized staphylococci may provide a renewed source of mastitis-producing staphylococcal regrowth.
Studies on the possible mechanism of antibiotic evasion of phagocytized staphylococci in mastitis treatment show that lysostaphin had been rejected as a candidate for destroying phagocytized staphylococci. Craven et al., 29 Research in Veterinary Science 57 (1980); Craven et al., 21 Antimicrobial Agents and Chemotherapy 618 (1982); Craven et al., 5 Comp. Immun. Microbial. Infect. Dis. 447 (1982)) Craven et al., 51 Journal of Dairy Research 513 (1984). In these experiments lysostaphin was used in vitro as a pretreatment to destroy extracellular staphylococci prior to exposing the phagocytized staphylococci to cloxacillin, gentamicin or lysostaphin. Craven et al.'s results strongly suggest that lysostaphin would have no effect on mastitis since intracellular staphylococci were still viable after 20 hours of incubation in a lysostaphin containing solution. 51 Journal of Dairy Research at 515-516, and Table 2.
Lysostaphin has also been reported to penetrate human monocytes. Since monocytes are a different cell type than PMNs, this human model is not likely to be applicable to the treatment of bovine mastitis (van den Broek et al., 21 Scand. J. Immunol 189 (1985)).
Lysostaphin has also been shown to be effective in the treatment of staphylococcal renal abscesses in mice, particularly when used in sequence with the administration of methicillin. Dixon et al., 41 Yale J. Biol. Med. 62 (1968).
In man lysostaphin has also been used as a therapeutic agent for treatment of chronic nasal staphylococcal infections (Quickel, Jr. et al., 22 Applied Microbiology 446 (1971)). In one case of a resistant staphylococcal infection, lysostaphin was given systemically (Stark et al., 291 Medical Intelligence 239 (1974)). In general, however, there has been great skepticism and reluctance in the medical and veterinary communities concerning the systemic administration of lysostaphin. Lysostaphin was considered to be too highly immunogenic to have general use for anything but topical applications.